Project Abstract Very few viruses are able to manifest as chronic infections in humans. The intrinsic innate immune response provides a first line of defense against invading viruses; however, in the case of chronic viral infections, these initial responses that were ineffective at controlling virus replication can then cause disease over many years due to chronic activation. In most organs, epithelial cells are some of the first cells to encounter viruses in the human body and innate immune responses in these cells are paramount to driving subsequent immune control. Interestingly, epithelial cells predominantly produce type III interferons (IFNs) in response to viral infection whereas immune cells produce Type II IFNs (?) and Type I IFNs (?/?) are produced by most cells in the body. The mechanism underlying cell type and tissue specific expression of the type III IFNs are unknown and likely involve regulation of epigenetics modifications, gene expression of pattern recognition receptors and associated signaling molecules. In drosophila, the fat body is the primary innate immune organ producing antimicrobial peptides in response to pathogens. The human liver, equivalent to the drosophila fat body in terms of function, utilizes Type III IFN responses to fight viral infection and likely possesses other unique properties with respect to innate immunity when compared to other organs. We have developed novel and exciting in vitro models that utilize primary epithelial cells from several organs that have intact innate immune responses when compared to immortalized or transformed cell lines. We and others have shown that these cell types are of critical importance in the development of disease since they directly detect components of viral pathogens. We therefore assert that primary cells are the optimal model to use for studies on innate immunity and we propose a novel approach to study innate immunity based on the innate immune pathways that we have demonstrated to be important for microbial pathogenesis. In addition, we are developing novel physiologic models incorporating primary epithelial cells, stem cell-derived epithelial cells, 3-dimensional chip and microfluidic-based platforms. The use of stem cell-derived cells would facilitate the identification of changes in gene expression, which occur during differentiation, that contribute to the unique innate immune system in epithelial cells. The specific goals of this program are to functionally characterize the innate immune response, including the production of Type III IFNs, to multiple viral pathogen associated patterns, including both DNA and RNA sensing pathways, and to elucidate the underlying molecular mechanisms through which innate immunity manifests in epithelial cells using sophisticated in vitro models. In addition, tissue specific and developmental expression of specific innate immune signaling components including, TLR3, STING and IRF7, will be addressed as a mechanism underlying tissue specific responses. Completion of these studies would offer the most in depth characterization of innate immunity in epithelial and other cell- types while improving our understanding of its contribution to human disease in multiple organs.